Dataset Information

Taking advantage of a high-throughput flow cytometer for the implementation of an ADCC assay for regulatory compliance.

ABSTRACT: Technological advances allowed the development of high-throughput instruments such as IntelliCyt iQue Screener PLUS®. Here, we took advantage of this technology to transfer a previously validated cytotoxicity assay. The evaluated parameters were cell permeability, caspase activation and phosphatidyl serine exposure. The assay was accurate (r2 = 0.90), precise (%CV ? 18.90) and specific. These results showed that this technology is suitable to be used in control quality environments. In addition, the automation provided a faster acquisition and analysis of data with precise and accurate results. This application could be implemented to evaluate another in vitro mechanism of action of different biotherapeutics.

SUBMITTER: Camacho-Sandoval R

PROVIDER: S-EPMC7193121 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Taking advantage of a high-throughput flow cytometer for the implementation of an ADCC assay for regulatory compliance.

Camacho-Sandoval Rosa R Jiménez-Uribe Alexis A Tenorio-Calvo Alejandra V AV López-Morales Carlos A CA Muñoz-García Leslie L Montes-Luna Alejandra A García-Xolalpa Héctor Leonardo HL Velasco-Velázquez Marco M Pavón Lenin L Pérez-Tapia Sonia Mayra SM Medina-Rivero Emilio E

Biotechnology reports (Amsterdam, Netherlands) 20200419

Technological advances allowed the development of high-throughput instruments such as IntelliCyt iQue Screener PLUS®. Here, we took advantage of this technology to transfer a previously validated cytotoxicity assay. The evaluated parameters were cell permeability, caspase activation and phosphatidyl serine exposure. The assay was accurate (r<sup>2</sup> = 0.90), precise (%CV ≤ 18.90) and specific. These results showed that this technology is suitable to be used in control quality environments. I ...[more]

PMID: 32373484

Similar Datasets

Project description:BackgroundThe production of stable and soluble proteins is one of the most important steps prior to structural and functional studies of biological importance. We investigated the parallel production in a medium throughput strategy of genes coding for proteins from various marine organisms, using protocols that involved recombinatorial cloning, protein expression screening and batch purification. This strategy was applied in order to respond to the need for post-genomic validation of the recent success of a large number of marine genomic projects. Indeed, the upcoming challenge is to go beyond the bioinformatic data, since the bias introduced through the genomes of the so called model organisms leads to numerous proteins of unknown function in the still unexplored world of the oceanic organisms.ResultsWe present here the results of expression tests for 192 targets using a 96-well plate format. Genes were PCR amplified and cloned in parallel into expression vectors pFO4 and pGEX-4T-1, in order to express proteins N-terminally fused to a six-histidine-tag and to a GST-tag, respectively. Small-scale expression and purification permitted isolation of 84 soluble proteins and 34 insoluble proteins, which could also be used in refolding assays. Selected examples of proteins expressed and purified to a larger scale are presented.ConclusionsThe objective of this program was to get around the bottlenecks of soluble, active protein expression and crystallization for post-genomic validation of a number of proteins that come from various marine organisms. Multiplying the constructions, vectors and targets treated in parallel is important for the success of a medium throughput strategy and considerably increases the chances to get rapid access to pure and soluble protein samples, needed for the subsequent biochemical characterizations. Our set up of a medium throughput strategy applied to genes from marine organisms had a mean success rate of 44% soluble protein expression from marine bacteria, archaea as well as eukaryotic organisms. This success rate compares favorably with other protein screening projects, particularly for eukaryotic proteins. Several purified targets have already formed the base for experiments aimed at post-genomic validation.

Dataset Information

Taking advantage of a high-throughput flow cytometer for the implementation of an ADCC assay for regulatory compliance.

Publications

Taking advantage of a high-throughput flow cytometer for the implementation of an ADCC assay for regulatory compliance.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets